Astaxanthin improves the development of the follicles and oocytes through alleviating oxidative stress induced by BPA in cultured follicles

This study is to investigate whether astaxanthin could alleviate the oxidative stress damages of follicles induced by BPA and improve the development of the cultured follicles and oocytes. Compared with BPA group, the survival rate, antrum formation rate, oocyte maturation rate and adherence area of the D8 and D10 follicles of the BPA+Asta group were significantly higher. The estrogen and progesterone in the culture medium of BPA+Asta group were significantly higher. PCNA in D8 and D10 granulosa cells and ERα in D10 granulosa cells of follicles in BPA+Asta group were significantly higher. The levels of malondialdehyde in the follicle culture medium, levels of ROS in the oocytes, the expression levels of caspase 3 and cathepsin B in the oocytes of the BPA+Asta group were significantly lower. However, the mitochondrial membrane potential, and the expression levels of antioxidant genes (CAT, SOD1 and SOD2) and anti-apoptotic gene Bcl-2 in the oocytes in the BPA+Asta group were significantly higher. Astaxanthin improves the development of follicles and oocytes through increasing the antioxidant capacity of follicles and oocytes, and relieving the BPA-induced oxidative stress during follicular development and oocyte maturation.


Results
Astaxanthin alleviates inhibitory effects of BPA on follicular development and oocyte maturation. Preantral follicles were cultured in vitro for 11 d. The developmental status of the follicles were observed on D2, D4, D6, D8 and D10 (Fig. 1). In the control group and DMSO group, on D2, the follicles begin to grow adherently, and the follicular theca cells grew outward. Subsequently, as the granulosa cells proliferated, the volume of the follicle gradually was increased. On D10, the follicle antrum was observed. At 14-16 h after adding hCG, mature follicles ovulated. In the BPA group, on D2, a few follicles did not grow adherently and died on the following days. From D6 to D10, the follicles were smaller than those in the control group and DMSO group. On D10, antrum could not be formed in a few follicles. In the BPA+Asta group, from D2 to D10, the development of the follicles was similar to that in the control group and DMSO group. The follicle development rates of these groups were analyzed and shown in Table 1. Compared with the control group, the survival rate, antrum formation rate and oocyte maturation rate of the BPA group were significantly decreased. The survival rate and antrum formation rate of the follicles, and the maturation rate of oocytes, in the BPA+Asta group, were significantly higher than the BPA group. The 0.2% DMSO showed no significant effects on the development rate of follicles at each stage. There was no significant difference in the ovulation rate of follicles among these groups. These results suggest that, astaxanthin could attenuate the inhibitory effects of BPA on follicular development and oocyte maturation.
Astaxanthin promotes granulosa cell proliferation. The adherence area of D2, D4, D6, D8 and D10 follicles were observed and analyzed. As shown in Fig. 2, compared with the control group, the adherence area of the D4, D6, D8 and D10 follicles was significantly reduced in the BPA group (P < 0.05). The adherence area of the D2, D4 and D6 follicles in the BPA+Asta group was not significantly different from the BPA group, while that of the D8 and D10 follicles in the BPA+Asta group was significantly larger than the BPA group (P < 0.05) (Fig. 2). The enlargement of the adherence area indicated the proliferation of the granulosa cells from the follicle. Astaxanthin increased the adherence area of the follicles on D8 and D10, indicating that it promoted the proliferation of the granulose cells from the follicles on D8 and D10.
Western blotting was performed to detect the expression of PCNA, which is an indicator of cell proliferation. As shown in Fig. 3, compared with the control group and the DMSO group, the PCNA expression levels in the D8 and D10 granulosa cells in the BPA group were significantly reduced (P < 0.05). The PCNA expression levels of D8 and D10 granulosa cells in the BPA+Asta group were significantly higher than the BPA group (P < 0.05), which was still significantly lower than the control group (P < 0.05) on D8 and D10. These results suggest that astaxanthin could increase the expression of PCNA in granulosa cells, thus promoting their proliferation.
Astaxanthin improves secretion of estrogen and progesterone and expression of ERα in granulosa cells. The culture medium of D10 follicles was collected separately, and the levels of estrogen and progesterone secreted by the follicles were measured by ELISA. As shown in Fig. 4, compared with the control group, the levels of estrogen and progesterone in the culture medium of the BPA group were significantly reduced (P < 0.05) ( Fig. 4A and B). The levels of estrogen and progesterone in the culture medium of the BPA+Asta group were higher than those in the BPA group (P < 0.05). These results suggest that astaxanthin increases the secretion of estrogen and progesterone in D10 follicular granulosa cells.
The granulosa cells of D10 follicles were collected separately for Western blotting analysis of ERα. As shown in Fig. 4C, compared with the control group, the expression level of ERα in the granulosa cells in the BPA group and BPA+Asta group was significantly reduced (P < 0.05) (Fig. 4C) www.nature.com/scientificreports/ granulosa cells in the BPA+Asta group was significantly higher than the BPA group (P < 0.05). These results suggest that astaxanthin stimulates the expression of ERa in granulosa cells in the late stage of follicular development.
Astaxanthin reduces malondialdehyde (MDA) and ROS levels in follicle culture medium and oocytes. MDA is a metabolite of membrane lipid peroxidation. The content of MDA in the culture medium reflects the damage degree to the cell membrane of follicle cells after oxidative stress, which is closely related to the developmental potential of the follicle 32 . In this study, the D10 follicle culture medium was collected and subjected to the MDA detection. As shown in Fig. 5, compared with the control group, the levels of MDA in the follicular culture medium of the BPA group and the BPA+Asta group were significantly increased (P < 0.05). After astaxanthin treatment, MDA level in the culture medium of the BPA+Asta group was significantly reduced than In the picture of ovulation, the arrows indicated the ovulated cumulus-oocyte complexes (CoCs); and in the picture of M II oocytes, the arrows indicated the first polar body excreted by mature oocytes. www.nature.com/scientificreports/ that of the BPA group (P < 0.05). These results suggest that astaxanthin protects cell membranes of the cultured follicles from lipid peroxidation through reducing the production of MDA by follicles. ROS staining was then performed on the oocytes discharged from D11. The stronger the green fluorescence in the oocytes, the higher the content of ROS (Fig. 6A). Statistically, the average fluorescence intensity of ROS in oocytes in the BPA group was significantly higher than the control group (P < 0.05) (Fig. 6B). The average fluorescence intensity of ROS in oocytes of the BPA+Asta group was significantly lower than the BPA group (P < 0.05) (Fig. 6B). These results suggest that astaxanthin reduces the production of ROS in oocytes.
Astaxanthin increases mitochondrial membrane potential of oocytes and changes antioxidant and apoptosis-related gene expression levels. JC-1 probe was used to detect the mitochondrial membrane potential of oocytes. The higher ratio of red fluorescence to green fluorescence indicated that the mitochondrial membrane potential was normal, and the cells were in a healthy state (Fig. 7A). The ratio of red to green fluorescence of oocytes in the BPA group was significantly lower than the control group (P < 0.05) (Fig. 7B). The ratios of red to green fluorescence of oocytes in the BPA+Asta group were significantly higher than the BPA group (P < 0.05), and there was no significant difference between BPA+Asta group and control group. These results suggest that astaxanthin increases the mitochondrial membrane potential.
Quantitative real-time PCR was used to detect the expression of antioxidant genes (CAT (catalase), SOD1 (superoxide dismutase 1), and SOD2 (superoxide dismutase 2)), apoptosis-related genes (caspase 3 and Bcl-2), and cathepsin B in oocytes (Fig. 8). Our results showed that the expression levels of antioxidant genes (CAT, SOD1, and SOD2) and the anti-apoptotic gene Bcl-2 in the oocytes of the BPA group were significantly lower than the control group (P < 0.05) (Fig. 9). On the other hand, the expression levels of caspase 3 and cathepsin B were significantly higher in BPA group than the control group (P < 0.05). Importantly, in BPA+Asta group, the expression levels of anti-oxidant genes (CAT, SOD1, and SOD2) and anti-apoptotic gene Bcl-2 were significantly higher than those in the BPA group, while the expression levels of caspase 3 and cathepsin B were significantly lower (P < 0.05). These results suggest that astaxanthin promotes the expression levels of antioxidant genes in oocytes and may inhibit oocyte apoptosis via regulating apoptosis-related genes.  www.nature.com/scientificreports/

Discussion
More and more studies have shown that BPA is an important environmental substance that affects human reproduction [33][34][35][36] . It can inhibit the proliferation of ovarian granulosa cells, reduce the expression of connexins, and inhibit the expansion of CoCs and affect the meiosis recovery and maturation of ovarian oocytes [33][34][35][36] . However, these studies were performed in granulosa cells, oocytes or CoCs in the ovary separately [34][35][36] , which cannot better simulate the exposure to BPA in vivo. In this study, preantral follicles were separated. They were cultured in vitro for 11 days and exposed to BPA during the entire process of follicular development. The in vitro culture system of preantral follicles better maintained the connections between oocytes, granulose cells and theca cells. The effects of BPA on the development of follicles, the proliferation of granulosa cells and the maturation of oocytes were investigated simultaneously.   www.nature.com/scientificreports/ Oxidative stress has been considered to be an important harmful factor affecting the development of follicles, oocytes and embryos [37][38][39] . As a metabolic and endocrine disruptor, BPA can disrupt the oxidative balance in cells through direct or indirect ways, such as increasing mediators of oxidation reaction, reducing the production of antioxidant enzymes, damaging the mitochondrial function, and inducing apoptosis 40,41 . It can reduce the expression levels of antioxidant enzymes SOD, CAT, glutathione reductase and glutathione peroxidase in rat liver and epididymal sperm, and increase the contents of hydrogen peroxide in cells and the occurrence of lipid peroxidation 42,43 . In addition, cathepsin B is a lysosomal cysteine protease of the papain family, which can induce cell apoptosis, extracellular matrix degradation and intracellular protein catabolism 44 . The activity of cathepsin B is negatively correlated with the quality of oocytes and embryos 44 . When cathepsin B activity is reduced, the embryonic cell apoptosis is decreased and the developmental capacity and quality of embryos is improved 45 . Consistently, our results showed that 25 μM BPA significantly increased the levels of MDA in follicular culture medium, decreased the mitochondrial membrane potential of oocytes, the expression of antioxidant genes (CAT, SOD1, and SOD2) and the anti-apoptotic gene Bcl-2, and increased the caspase 3 and cathepsin B expression levels. These findings suggest that BPA could induce the lipid peroxidation in the follicles, weaken the anti-oxidation ability of the oocytes, induce apoptosis, and reduce the quality of the oocytes.
Astaxanthin is a strong fat-soluble antioxidant that can reduce lipid peroxidation and DNA damage in oocytes and embryos 28 . It is found that astaxanthin significantly increased the expression levels of antioxidant gene GPX4 in aging pig oocytes, the levels of reduced glutathione in oocytes, and the mRNA levels of BCL2L1 and SURVIVIN, but significantly reduced the cathepsin B activity and caspase-3 mRNA levels, thereby improving the developmental ability of aging oocytes and parthenogenetic embryos 30 . For frozen pig oocytes, astaxanthin could significantly increase the levels of glutathione and mitochondrial activity in the cells, thus improving the anti-oxidation and development of the frozen oocytes 46 . In this study, astaxanthin was used to alleviate the oxidative damages of BPA to follicular development. Our results showed that the mitochondrial membrane potential, the expression levels of antioxidant genes (CAT, SOD1, and SOD2) and anti-apoptotic gene Bcl-2 in oocytes of the BPA+Asta group were significantly increased. The levels of MDA in the culture medium and the expression levels of caspase 3 and cathepsin B in oocytes were significantly reduced. Thus, we suppose that astaxanthin could improve the antioxidant capacity of oocytes, inhibit oocyte apoptosis, and alleviate the damages of BPA to the development of oocytes. In addition, astaxanthin significantly increased the survival rate, antrum formation rate, and oocyte maturation rate of follicles, and increased the expression levels of PCNA in granulosa cells in the late stage of follicular development, promoting the proliferation of granulosa cells.
Oxidative stress is mainly caused by excessive ROS production or defects in the antioxidant mechanism in the cells 47 . ROS would react with intracellular macromolecular substance, such as lipids, proteins and DNA, causing intracellular enzyme inactivation, destruction of cell membrane integrity, abnormal mitochondrial function, and DNA fragmentation 47 . ROS can also block and delay the development of preimplantation embryos, and reduce the in vitro development rate of preantral follicles 31 . BPA can significantly increase the lipid peroxidation and ROS production of ovarian tissue, and affect the normal development of follicles and oocytes 5 . BPA can also www.nature.com/scientificreports/ significantly increase the ROS levels in pig embryos, causing intracellular cytochrome C release, mitochondrial and DNA damages, and apoptosis 48 . It is shown that antioxidants can reduce the production of ROS 28 , increase the expression levels of antioxidant genes in cells, and improve the maturation, fertilization and early embryonic development capabilities of oocytes 49 . Jia et al. 30 reported that 2.5 μM astaxanthin significantly reduced the production of ROS in oocytes and improved the quality of oocytes 30 . In this study, we have obtained similar results, which showed that the BPA treatment significantly increased the ROS levels in oocytes and reduced the maturation rate of oocytes. On the other hand, astaxanthin significantly reduced the production of ROS in oocytes, significantly increased the maturation rate of oocytes, and significantly relieved the oxidative stress damage in oocytes caused by BPA. The whole process of follicular development is accompanied by the continuous proliferation of granulosa cells, and the steroid hormones synthesized and secreted by granulosa cells and theca cells further stimulate the development of follicles 50 . BPA is reported to inhibit the proliferation of granulosa cells, interfere with the synthesis of steroid hormones, and interfere with the follicular development 50 . Since the volume of oocytes hardly changes during the development of preantral follicles with the diameter of 110-130 μm 50 , the adherence area of follicles may reflect the proliferation of granulosa cells. Additionally, PCNA is involved in regulating DNA synthesis and is an important indicator of cell proliferation 50 . In this study, BPA was added to the mouse preantral follicle www.nature.com/scientificreports/ culture medium, and our results showed that the adherence area of the follicles in the BPA group was significantly reduced from D4, and the PCNA expression levels of D8 and D10 granulosa cells were also significantly lower than the control group. This suggests that BPA may inhibit the proliferation of granulosa cells. Zhou et al. 51 found that in the in vitro culture of granulosa cells, BPA inhibited the proliferation of rat follicular theca cells and granulosa cells, and reduced the secretion of estrogen and progesterone. Abdel-Ghani et al. 31 showed that astaxanthin (500 μM) significantly increased the synthesis and secretion of estradiol in follicles, and decreased the synthesis and secretion of progesterone. Kamada et al. 52 have found that low concentration (0.1-10 nM) of astaxanthin could increase the synthesis of progesterone in the luteal cells, while high concentration (1000 nM) of astaxanthin could inhibit the synthesis of progesterone. Therefore, we speculate that the effects of astaxanthin on progesterone synthesis may not be caused by the antioxidant effect. In this study, 2.5 nM astaxanthin was added into the in vitro culture medium of preantral follicles, which significantly increased the secretion of estrogen and progesterone in the follicles, and increased the expression levels of ERα in the granulosa cells, and the estrogen secreted by the follicles. The secreted estrogen would bind to the ER of the follicle to further stimulate the production of estrogen 53 . Astaxanthin significantly alleviated the interference of BPA on the synthesis of follicular steroid hormones. However, whether it works through the mechanism of alleviating antioxidant effects is still unclear, and further studies are warranted.
In conclusion, our results showed that astaxanthin improved the antioxidant capacity of oocytes by increasing the expression levels of antioxidant genes in oocytes, reduced the lipid peroxidation of follicles and the production of ROS, and protected oocytes against BPA-induced damages of the cell mitochondrial membrane potential, therefore promoting the proliferation of granulosa cells, improving the development rate of follicles and the maturation rate of oocytes, and significantly alleviating the oxidative stress damages to the follicles. Astaxanthin also increased the secretion of estrogen and progesterone in follicles, and relieved the inhibitory effects of BPA on the synthesis of steroid hormones. However, it was still necessary to further clarify the relationship between hormone synthesis and the antioxidant activity of astaxanthin. Therefore, as a high-efficiency antioxidant, astaxanthin is expected to become an antioxidant drug for the female reproductive system, protecting germ cells from oxidative stress damage and preventing premature ovarian failure and other diseases.

Methods
Study animals. The   www.nature.com/scientificreports/ were performed in accordance with relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines.
Preparation of BPA and astaxanthin. DMSO (D2650; Sigma, St. Louis, MO, USA) was used to dissolve BPA (239658; Sigma) and astaxanthin (SML0982; Sigma). BPA and astaxanthin stock solutions at 25 mmol/L and 2.5 μmol/L were prepared. Before use, 1 µL BPA or astaxanthin stock solution was added into 1 mL in vitro culture medium for follicle to make the working concentration of 25 µmol/L or 2.5 nmol/L. Previous studies have found that 25 µmol/L BPA has germ cell toxicity 44 , while 2.5 nmol/L astaxanthin has significant antioxidant activity 54 . In the solvent group, 2 μL DMSO was added into 1 mL in vitro culture medium for follicle to make the final concentration of 0.2%.
The in vitro culture and treatment of mouse preantral follicles. The in vitro culture of mouse preantral follicles was performed according to a previous method published by Liu et al. 55 Briefly, the 14-day Kunming female mice were sacrificed by cervical dislocation. Their bilateral ovaries were quickly removed to L-15 working solution (containing 10% FBS) (11415114; Gibco, GrandIsland, NY, USA). A 26G needle was used to mechanically isolate the follicles. The preantral follicles were selected according to the Pedersen and Peter's grading criteria for grade 4 or 5a follicles with a diameter of 110-130 μm 56 . Namely, the preantral follicles consisted of a round zona pellucida-wrapped oocyte in the middle of the follicle, 2-4 layers of granulosa cells, intact basement membrane and several theca cells attached to the basement membrane. These follicles are growing follicles. The selected preantral follicle was placed in a droplet of α-MEM culture medium (12571063; Gibco), which contained 5% FBS (10091155; Gibco), 1% ITS (41400045; Gibco) and 0.1 IU/ml r-FSH; Gonal-F, Serono), and cultured in a 37 °C, 5% CO 2 incubator, for 10 days.
In the BPA group, 25 µmol/L BPA was added into the culture medium, and in the DMSO group, 0.2% DMSO was added. In the BPA+Asta group, 25 µmol/L BPA and 2.5 nmol/L astaxanthin was added into the culture medium. The medium was semi-quantitatively replaced every other day. The supernatant was collected and stored at − 20 °C for further analysis. The control group was untreated. The growth of follicles on day 0 (D0), day 2 (D2), day 4 (D4), day 6 (D6), day 8 (D8) and day 10 (D10) was observed and recorded, with the cellSens microscopic image software of the Olympus IX-83 microscope. On D2, the follicles that began to grow adherently were defined as survival follicles. The survival rate of the cultured follicles was calculated as the ratio of survival follicles to total cultured follicles. On D10, the antrum was formed in the follicle. The antrum formation rate was defined as the ratio of the follicles with antrum to the survival follicles. On D10, the culture medium containing 2.5 U/ml hCG (Livzon Pharmaceutical Factory) was added. After 14-16 h, mature follicles ruptured and ovulated naturally. The ovulation rate was defined as the ratio of the follicles that ovulated to the follicles with antrum. The ovulated CoCs were denuded. MII oocytes with the first polar body were mature oocytes. Maturation rate was defined as the ratio of the mature oocytes to the total denuded oocytes. The Image J software was used to analyze the adherence area of follicles on the petri dish, and 10 follicles were analyzed in each group.
ELISA. The levels of estrogen and progesterone in D10 follicle culture medium were detected with ELISA kits (BPE20376 and BPE20381; Shanghai Lengton Bioscience Co., Ltd., Shanghai, China), according to the kit instructions. The absorbance value was measured at 450 nm using a SpectraMax Absorbance Reader (Molecular Devices).
Western blotting analysis. In each group, granulosa cells were obtained from 40 follicles on Day 8 or 10 of the in vitro culture. Protein was extracted from granulosa cells with RIPA strong lysate (containing 1% PMSF) (P0013B and P1006; Beyotime) and separated by 12% SDS-PAGE. Protein was transferred onto the PVDF membrane, which was then blocked with 5% skimmed milk for 1 h. Then the membrane was treated with primary antibodies against proliferating cell nuclear antigen (PCNA, ab92552: Abcam, Cambridge, MA, USA), estrogen receptor α (ERα) (ab32063; Abcam) or β-actin (ab8226; Abcam) at 4 °C overnight. After washing with PBST, the membrane was incubated with HRP-conjugated goat anti-rabbit secondary antibody (31210; Thermo Scientific Pierce) at room temperature for 2 h. Color development was performed with the enhanced chemiluminescence. The protein bands were scanned using the ChemiDOC XRS + imaging systems (Bio-Rad Laboratories, Hercules, CA, USA). β-actin was used as an internal control for protein loading. Image J image analysis software was used to analyze the relative expression levels of PCNA and ER-α based on the density of β-actin. The original images of full-length blots are in the Supplementary Information. MDA content determination. MDA has been widely used as an indicator of lipid peroxidation 32 . It can react with thiobarbituric acid (TBA) at higher temperature and acidic environment to form a red MDA-TBA adduct, which could be detected by colorimetry 57 . The Lipid Peroxidation MDA Assay Kit (S0131S; Beyotime) was used to determine the contents of MDA in D10 follicle culture medium. The specific method was as follows 57 : D10 follicle culture medium frozen at − 20 °C was thawed, and centrifuged at 1600 × g at 4° C for Mitochondrial membrane potential detection with JC-1. The JC-1 probe was used to detect the mitochondrial membrane potential according to previously published methods 59,60 . Briefly, after washing with MII medium for 3 times, the oocytes were placed in a culture medium, containing 0.5 µmol/L JC-1 (Invitrogen, Grand Island, NY, USA) in a 37 °C, 5% CO 2 incubator for 30 min. JC-1 forms J-aggregates and produces red fluorescence under normal mitochondrial membrane potential. When there is cell apoptosis, the mitochondrial membrane potential would be decreased or even lost, and JC-1 exists in J-monomers and produces green fluorescence. Fluorescence was captured with the the cellSens microscopic image software using the Olympus IX-83 fluorescence microscope. The ratio of red and green fluorescence intensities of oocytes reflected the mitochondrial membrane potential. Totally 10 oocytes were analyzed from each group.
Quantitative real-time PCR. Totally, 60 oocytes were analyzed by quantitative real-time PCR. Total RNA was extracted from oocytes using the Rneasy Micro Kit (Qiagen, Hilden, Germany). Reverse transcription was performed to obtain cDNA in a 20 μL reverse transcription system, consisting of 1 μL random primers, Statistical analysis. Data were expressed as mean ± SD. The SPSS 17.0 statistical software was used for statistical analysis. Data were analyzed with the one-way ANOVA and the LSD post doc test. P < 0.05 was considered to be statistically significant.
Ethics approval. All the animal experimental procedures were approved by the ethics committee of the Jilin Medical College. All experiments were performed in accordance with relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.  AGC ATG GGT TCC ACG TCC ATCA  ACC GTC CTT TCC AGC AGT CACA  58   SOD2  ATA ATG TTG TGT CGG GCG GCGT  TCG GTG GCG TTG AGA TTG TTC ACG 60   Caspase 3  TGG CAT TGA GAC AGA CAG TGGGA  TGC GCG TAC AGC TTC AGC AT  57   Cathepsin B  TGG CAA GAT TTG GAC GAC TGG ACC ACA GCA GGC ACT ACA AAC CGCA  60 Bcl-2 ACC GTC GTG ACT TCG CAG AGAT TGT GCA GAT GCC GGT TCA GGTA 58 β-actin TGT TAC CAA CTG GGA CGA CA CTG GGT CA TCT TTT CAC GGT 58